Significant Increase In Cell Death Research Articles

Autophagic Cell Death and Apoptosis Jointly Mediate Cisatracurium Besylate-Induced Cell Injury.

Cisatracurium besylate is an ideal non-depolarizing muscle relaxant which is widely used in clinical application. However, some studies have suggested that cisatracurium besylate can affect cell proliferation. Moreover, its specific mechanism of action remains unclear. Here, we found that the number of GFP-LC3 (green fluoresent protein-light chain 3) positive autophagosomes and the rate of mitochondria fracture both increased significantly in drug-treated GFP-LC3 and MitoDsRed stable HeLa cells. Moreover, cisatracurium promoted the co-localization of LC3 and mitochondria and induced formation of autolysosomes. Levels of mitochondrial proteins decreased, which were reversed by the lysosome inhibitor Bafinomycin A1. Similar results with evidence of dose-dependent effects were found in both HeLa and Human Umbilical Vein Endothelial Cells (HUVECs). Cisatracurium lowered HUVEC viability to 0.16 (OD490) at 100 µM and to 0.05 (OD490) after 48 h in vitro; it increased the cell death rate to 56% at 100 µM and to 60% after 24 h in a concentration- and time-dependent manner (p < 0.01). Cell proliferation decreased significantly by four fold in Atg5 WT (wildtype) MEF (mouse embryonic fibroblast) (p < 0.01) but was unaffected in Atg5 KO (Knockout) MEF, even upon treatment with a high dose of cisatracurium. Cisatracurium induced significant increase in cell death of wild-type MEFs even in the presence of the apoptosis inhibitor zVAD. Thus, we conclude that activation of both the autophagic cell death and cell apoptosis pathways contributes to cisatracurium-mediated cell injury.

Effects of excess succinate on cardiomyocyte mitochondrial H2O2 production and cell death

IntroductionDuring myocardial infarction injury, reactive oxygen species (ROS) generation in the first few minutes of reperfusion has been shown to cause significant cellular dysfunction and death of the myocardium. Recent studies suggest ROS generation during reperfusion is due to accumulation of intermediate citric acid cycle metabolites, specifically succinate accumulation. Additionally, previous studies suggest that an increase in fumarate accompanied by an increase in succinate is cardioprotective. In this study, we examined the effect of excess succinate on H2O2 production and cell death. We hypothesized that excess succinate increases H2O2 production through reverse electron transport in mitochondria isolated from Sprague‐Dawley rat hearts and increases cell death in induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) and that addition of fumarate would abolish this phenotype.MethodsThe left ventricles of Sprague Dawley male rats were collected and minced. Mitochondria were isolated using differential centrifugation. Mitochondria were exposed to 5 mM succinate with or without 1μM rotenone (a complex I inhibitor). H2O2 production was measured using an Amplex Red assay. Human iPSCs were cultured to confluency and differentiated into CMs. Contracting cells were immunostained for cardiac specific markers to confirm &gt;90% purity of CMs. CMs were exposed to 5 mM dimethyl succinate with or without 5 mM dimethyl fumarate for 1 hour followed by 24 hours of reperfusion in basal media. Cell viability was measured using propidium iodide staining.ResultsExcess succinate caused a significant increase in H2O2 production in mitochondria isolated from Sprague Dawley rat hearts. This effect was abolished with addition of rotenone, showing that H2O2 production was occurring through reverse electron transport. Dimethyl succinate also caused significant cell death in iPSC‐derived CMs. Dimethyl fumarate with dimethyl succinate did not abolish increased cell death.ConclusionsWe have confirmed that excess succinate causes a significant increase in H2O2 production through reverse electron transport in mitochondria isolated from Sprague Dawley rat hearts and a significant increase in cell death in iPSC‐derived CMs. Fumarate in addition to excess succinate did not have a cardioprotective effect in iPSC‐CMs. However, this may be a toxic effect rather than a metabolic effect. Further experiments detailing the mechanism by which this occurs may reveal new therapeutic targets to reduce the detriment of ROS production from ischemia/reperfusion injury.Support or Funding InformationNIH/NIGMS Program Project Grant 2P01GM066730‐11Program Director: Zeljko J. Bosnjak “Mechanisms of Anesthetic Cardioprotection”Project II: “MicroRNA in Anesthetic Cardioprotection”

Fipronil induces lung inflammation in vivo and cell death in vitro.

BackgroundFipronil is an insecticide that acts at the gamma-aminobutyric acid receptor and glutamate-gated chloride channels in the central nervous systems of target organisms. The use of fipronil is increasing across the globe. Presently, very little data exist on the potential impact of exposure to fipronil on the lungs.MethodsWe studied effects of intranasal (N = 8) and oral (N = 8) treatment with fipronil (10 mg/kg) on lungs of mice. Control mice were given groundnut oil orally (N = 7) or ethanol intranasally (N = 7) as these were the vehicles for respective treatments.ResultsHematoxylin-eosin stained lung sections showed normal histology in the control lungs compared to the thickened alveolar septa, disruption of the airways epithelium and damage to vascular endothelium in the intranasal and the oral groups. Mice exposed to fipronil either orally or intranasally showed increased von Willebrand factor staining in the endothelium and septal capillaries. Compared to the control mice, TLR4 expression in airway epithelium was increased in mice treated intranasally but not orally with fipronil. Oral fipronil reduced TLR9 staining in the airway epithelium but intranasal exposure caused intense staining in the alveolar septa and airway epithelium. There were higher numbers of TLR4 positive cells in alveolar septa in lungs of mice treated intranasally (P = 0.010) compared to the respective control and orally treated mice but no significant differences between treatments for TLR9 positive stained cells (P = 0.226). The U937 macrophage cells exposed to fipronil at concentrations of 0.29 μm to 5.72 μm/ml over 3- or 24-hour showed significant increase in cell death at higher concentrations of fipronil (P < 0.0001). Western blots revealed no effect of fipronil on TLR4 (P = 0.49) or TLR9 (P = 0.94) expression on macrophage cell line.ConclusionWhile both oral or intranasal fipronil treatments induced signs of lung inflammation, the number TLR4-positive septal cells was increased only following intranasal treatment. Fipronil causes macrophage cell death without altering TLR4 and TLR9 expression in vitro.

Molecular distribution and toxicity assessment of praseodymium by Spirodela polyrrhiza

Aquatic macrophytes are known to accumulate and bioconcentrate metals. In this study, the physiological, biochemical, and ultrastructural responses of Spirodela polyrrhiza to elevated concentrations of praseodymium (Pr), ranging from 0 to 60μM, were investigated over 20 d exposure. The results showed that the accumulation of Pr in S. polyrrhiza occurred in a concentration-dependent manner. The accumulation of Pr in biomacromolecules decreased in the order of cellulose and pectin (65–69%), crude proteins (18–25%), crude polysaccharides (6–10%), crude lipids (3%–4%). Significant increases in malondialdehyde (MDA), and decreases in photosynthetic pigment, soluble protein, and unsaturated fatty acids showed that Pr induced oxidative stress. Inhibitory effects on photosystem II and the degradation of the reaction center proteins D1 and D2 were revealed by chlorophyll a fluorescence transients, immunoblotting, and damage to chloroplast ultrastructure. Significant increases in cell death were observed in Pr-treated plants. However, S. polyrrhiza can combat Pr induced oxidative injury by activating various enzymatic and non-enzymatic antioxidants. These results will improve understanding of the biological consequences of rare earth elements (REEs) contamination, particularly in aquatic bodies.

Protein DJ-1 and its anti-oxidative stress function play an important role in renal cell mediated response to profibrotic agents.

In the pathogenesis of renal fibrosis, oxidative stress (OS) enhances the production of reactive oxygen species (ROS) leading to sustained cell growth, inflammation, excessive tissue remodelling and accumulation, which results in the development and acceleration of renal damage. In our previous work (Eltoweissy et al., 2011) we established protein DJ-1 (PARK7) as an important ROS scavenger and key player in renal cell response to OS. In the present study we investigated the impact of profibrogenic agonists on DJ-1 and shed light on the role of this protein in renal fibrosis. Treatment of renal fibroblasts and epithelial cells with the profibrogenic agonist ANG II or PDGF resulted in a significant up-regulation of DJ-1 expression parallel to an increase in the expression of fibrosis markers. Monitoring of DJ-1 expression in kidney extract and tissue sections from a renal fibrosis mouse model (Col4a3-deficient) revealed a disease grade dependent regulation of the protein. Overexpression of DJ-1 prompted cell resistance to OS in both fibroblasts and epithelial cells. Furthermore overexpression of DJ-1, involved in ROS scavenging, in which glutamic acid 18 (E18) is mutated to either to aspartic acid (D) or glutamine (Q) resulted in a significant increase in cell death under OS in the case of E18D mutation, whereas E18Q mutation did not impact significantly the cell response to OS, revealing the importance of the acidic group for the ROS scavenging activity of the DJ-1 protein more than the nature of the amino acid itself. Affinity precipitation of interaction partners of DJ-1 and its mutants revealed an important role of annexin A1 and A5 in the mechanism of action of DJ-1 in anti-oxidative stress response.

Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival.

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection.

S6K1 inhibition enhances the apoptotic cell death of breast cancer cells in response to Bcl-2/Bcl-xL inhibition by the downregulation of survivin.

Breast cancer cells possess a deregulated apoptotic pathway with increased expression levels of anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins and ribosomal S6 kinase 1 (S6K1) protein activity. Therefore, combined interference of anti-apoptotic Bcl-2 family and S6K1 protein expression may be a reasonable therapeutic strategy for the treatment of patients with breast cancer. In the present study, it was identified that the administration of a combination of ABT263 [navitoclax; a Bcl-2/Bcl-extra large (Bcl-xL) inhibitor] and PF4708671 (an S6K1 inhibitor) markedly increased apoptotic cell death in the BT474 breast cancer cells compared with the administration of either agent alone. Furthermore, the downregulation of Bcl-2/Bcl-xL and S6K1 with small interfering RNA induced a significant increase in cell death compared with RNA interference of either agent alone. Notably, combination treatment with ABT263 and PF4708671 decreased the expression level of survivin protein, with this ectopic expression of survivin attenuating cell death. Thus, the present study determined that the combined inhibition of Bcl-2/Bcl-xL and S6K1 may be a good strategy for treating patients with breast cancer.

DT-Diaphorase Prevents Aminochrome-Induced Alpha-Synuclein Oligomer Formation and Neurotoxicity.

It was reported that aminochrome induces the formation of alpha synuclein (SNCA) oligomers during dopamine oxidation. We found that DT-diaphorase (NQO1) prevents the formation of SNCA oligomers in the presence of aminochrome determined by Western blot, transmission electron microscopy, circular dichroism, and thioflavin T fluorescence, suggesting a protective role of NQO1 by preventing the formation of SNCA oligomers in dopaminergic neurons. In order to test NQO1 protective role in SNCA neurotoxicity in cellular model, we overexpressed SNCA in both RCSN-3 cells (wild-type) and RCSN-3Nq7 cells, which have constitutive expression of a siRNA against NQO1. The expression of SNCA in RCSN-3SNCA and RCSN-3Nq7SNCA cells increased 4.2- and 4.4-fold, respectively. The overexpression of SNCA in RCSN-3Nq7SNCA cells induces a significant increase in cell death of 2.8- and 3.2-fold when they were incubated with 50 and 70 µM aminochrome, respectively. The cell death was found to be of apoptotic character determined by annexin/propidium iodide technique with flow cytometry and DNA laddering. A Western blot demonstrated that SNCA in RCSN-3SNCA is only found in monomer form both in the presence of 20 µM aminochrome or cell culture medium contrasting with RCSN-3Nq7SNCA cells where the majority SNCA is found as oligomer. The antioligomer compound scyllo-inositol induced a significant decrease in aminochrome-induced cell death in RCSN-3Nq7SNCA cells in comparison to cells incubated in the absence of scyllo-inositol. Our results suggest that NQO1 seems to play an important role in the prevention of aminochrome-induced SNCA oligomer formation and SNCA oligomers neurotoxicity in dopaminergic neurons.

Cardiovascular effects in vitro of a polysaccharide from Salvia miltiorrhiza.

A polysaccharide (SMP1) was isolated from the roots of Salvia miltiorrhiza. This study is designed to investigate whether SMP1 prevents H9c2 cells from hydrogen peroxide (H2O2)-induced apoptosis. The present study showed that exposure of H9c2 cells to 100mM H2O2 for 24h caused a significant increase in cell death and apoptosis, but pretreatment with SMP1 eliminated H2O2-induced apoptotic cell death. Furthermore, pretreatment with SMP1 significantly prevented the mitochondria disruption, cytochrome c release, the rise of the ratio between proapoptotic Bax and antiapoptotic Bcl-2 protein expression, and caspase-3 activation in H9c2 cells upon H2O2 stimulation. Moreover, the decline of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities together with the elevation of malondialdehyde (MDA) in PC12 cells exposed to H2O2 were remarkably reversed to normal levels by pretreatment with SMP1. These results suggest that SMP1 protects H9c2 cells from H2O2-induced apoptosis through inhibition of mitochondrial dysfunction, inactivation of caspase-3 cascade and enhancement of antioxidant capacity.

Decreasing Mitochondrial Fission Prevents Cholestatic Liver Injury

Mitochondria frequently change their shape through fission and fusion in response to physiological stimuli as well as pathological insults. Disrupted mitochondrial morphology has been observed in cholestatic liver disease. However, the role of mitochondrial shape change in cholestasis is not defined. In this study, using in vitro and in vivo models of bile acid-induced liver injury, we investigated the contribution of mitochondrial morphology to the pathogenesis of cholestatic liver disease. We found that the toxic bile salt glycochenodeoxycholate (GCDC) rapidly fragmented mitochondria, both in primary mouse hepatocytes and in the bile transporter-expressing hepatic cell line McNtcp.24, leading to a significant increase in cell death. GCDC-induced mitochondrial fragmentation was associated with an increase in reactive oxygen species (ROS) levels. We found that preventing mitochondrial fragmentation in GCDC by inhibiting mitochondrial fission significantly decreased not only ROS levels but also cell death. We also induced cholestasis in mouse livers via common bile duct ligation. Using a transgenic mouse model inducibly expressing a dominant-negative fission mutant specifically in the liver, we demonstrated that decreasing mitochondrial fission substantially diminished ROS levels, liver injury, and fibrosis under cholestatic conditions. Taken together, our results provide new evidence that controlling mitochondrial fission is an effective strategy for ameliorating cholestatic liver injury.

Progesterone receptor membrane component 1 deficiency attenuates growth while promoting chemosensitivity of human endometrial xenograft tumors

Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A lentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4. To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 µM), doxorubicin (Dox, 2 µg/ml), or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dox-induced cell death. This response to P4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitoneal inoculation of immunocompromised NOD/SCID and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paclitaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (50 mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1-intact control tumors, suggesting that PGRMC1 promotes tumor cell viability during chemotherapeutic stress. In sum, these in vitro and in vivo findings demonstrate that PGRMC1 plays a prominent role in the growth and chemoresistance of human endometrial tumors.

Abstract 2780: Tyro3 and Axl receptors tyrosine kinase as potential therapeutic targets in leiomyosarcoma

Abstract Leiomyosarcoma (LMS) represents 15% of adult sarcomas. Once metastatic, curative options are limited and innovative strategies are needed. TYRO3 and AXL proto-oncogenes belong to TAM family (Tyro3/Axl/Mer) of tyrosine kinase receptors, with oncogenic properties that are often overexpressed or activated in various malignancies. Using kinexus phosphoprotein microarrays (KAM-1.1) and western blot analyses, we evidenced that Tyro3 and Axl are overexpressed in LMS along with Gas6, their ligand. Interestingly LMS samples harbouring high levels of Gas6 and its receptors also presented AKT phosphorylation on S473 residue keeping with previous studies that highlighted AKT- mTOR activation in leiomyosarcomas. Based on these premises, we asked whether Tyro3-Axl axis may drive LMS proliferation and could represent potential targets in these tumors. The overexpression of Tyro3 was found to be associated with a specific miRNA signature, notably with an overexpression of miR22 in LMS tumors. Furthermore Tyro3 and Axl were found overexpressed in a panel of 6 LMS cell lines (SK-LMS-1 from ATCC and IB112, IB118, IB133, IB134 and IB136 from Institut Bergonié), compared with normal human smooth muscle. Tyro3 or Axl knockdown via shRNA retroviral vector reduced cell proliferation by up to 50 and 64% respectively and decreased soft agar colony-forming ability. Crizotinib and foretinib are two tyrosine kinase inhibitors (TKI) targeting Met and Axl. Treatment of LMS cell lines with both TKIs resulted in a significant increase in cell death, with IC50 values ranging from 3.6 to 14.6µM and 0.7 to 1.8µM, respectively. Annexin V and PI staining revealed that cell death was mainly due to apoptosis. Flow cytometric cell cycle analysis demonstrates that both TKIs altered cell cycle phase distribution. A sub-G1 phase accumulation was observed for one of the cell lines; meanwhile the other cell lines presented a G2 arrest and 4n chromosomes accumulation accompanied by an increase in cell and nucleus sizes. Only foretinib induced nuclear fragmentation consistent with late apoptosis. Pharmacological inhibition also reduced migration and colony formation in soft agar. Drug combinations with an AKT inhibitor led to significant increase in growth inhibition in all tested cell lines. Crizotinib and foretinib reduced activation of Tyro3 or Axl-mediated downstream signaling including MAPK/ERK, PI3K/AKT along with an inhibition of Axl phosphorylation in a dose dependent manner. By immunoprecipitation we observed also a reduction in Tyro3 phosphorylation, following exposure to both agents, showing that Tyro3 is a new target for those drugs. IHC analysis of Tyro3, Axl and Gas6 expression in a cohort of LMS patient is ongoing. In vivo analysis wil be performed. In conclusion these results strongly suggest that Tyro3 and Axl are relevant therapeutic targets for LMS treatment and will be investigated in a proof of concept clinical trial. Citation Format: Carmela Dantas-Barbosa, François Le Loarer, Marta Mendiola, Isabelle Treilleux, Frederic Chibon, Hyba El Sayadi, Jean Michel Coindre, Laurent Alberti, Jean-Yves Blay. Tyro3 and Axl receptors tyrosine kinase as potential therapeutic targets in leiomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2780. doi:10.1158/1538-7445.AM2014-2780

Abstract 3096: Obinutuzumab (GA101) significantly induces antiproliferative effects and programmed cell death, and significantly downregulates cell signaling pathways in primary mediastinal B-cell lymphoma (PMBL): Obinutuzumab may be a future potential targeted agent for treatment of PMBL

Abstract BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) is a rare form of non-Hodgkin Lymphoma (NHL) representing 2% of mature B-cell NHL in patients ≤ 18 years of age (Lones/Cairo et al., JCO, 2000). PMBL is classified as a distinct mature B-Cell lymphoma and is considered midway in the biologic spectrum between Diffuse Large B-Cell Lymphoma (DLBCL) and classical HL (Abramson et al., Blood, 2005). We have recently reported a significant decrease in EFS among children and adolescent PMBL patients compared with other stage III non-PMBL pediatric DLBCL patients following FAB/LMB 96 therapy, suggesting that children and adolescent PMBL may be an inherently different B-NHL (Gerrard/Cairo et al., Blood, 2013). Since 98% of PMBL express CD20, targeting the CD20 receptor with a CD20 antibody is of high clinical interest. Obinutuzumab (GA101) is a novel, anti-CD20 targeted monoclonal antibody recognizing a unique CD20 type II epitope and has been demonstrated to have efficacy in reducing tumor size and improving survival in other B-NHL xenograft models (Mössner et al., Blood, 2010). OBJECTIVES: We hypothesize that obinutuzumab may be a future potential targeted agent for the treatment of PMBL, and therefore, we investigated the effect of obinutuzumab on cell proliferation, programmed cell death, and cell signaling pathways in PMBL. METHODS: CD20+ PMBL, Karpas-1106P cells (Karpas) were obtained from the DSMZ, Germany and obinutuzumab was generously provided by C. Klein, Roche. Cell proliferation and apoptotic rate were assessed using MTS and FACS analysis, respectively. Statistical significance was determined by one-tailed Student's t-test. RESULTS: There was a significant decrease of cell proliferation in obinutuzumab-treated Karpas vs PBS-treated Karpas (1.000 ± 0.000) at 48 hours with 1ug/ml (0.723 ± 0.005, p=0.0007), 10ug/ml (0.688 ± 0.025, p=0.0033), and 20ug/ml (0.627 ± 0.042, p=0.0092) obinutuzumab treatment. There was a significant increase in cell death in Karpas following 10ug/ml obinutuzumab treatment (37.790 ± 10.096, p=0.018) as compared to IgG isotope (1.040 ± 0.834, p=0.340) and PBS control (1.190 ± 0.762) at 48 hours by FACS analysis. We also observed significant decreases in the phosphorylation of Stat6 (0.730 ± 0.005, p=0.0001), Akt (0.691 ± 0.002, p&amp;lt;0.0001), IkBa (0.706 ± 0.006, p=0.0002) and Erk (0.578 ± 0.001, p&amp;lt;0.0001) in 10ug/ml obinutuzumab-treated Karpas as compared to control (1.000 ± 0.000). CONCLUSIONS: Obinutuzumab significantly induced antiproliferative effects and cell death in PMBL and may be a future potential targeted agent for the treatment of PMBL. The effect(s) of obinutuzumab will be evaluated in vivo in a NOD/SCID PMBL xenograft mouse model. Citation Format: Changhong Yin, Timmy O'Connell, Janet Ayello, Carmella van de Ven, Sanghoon Lee, Mitchell Cairo. Obinutuzumab (GA101) significantly induces antiproliferative effects and programmed cell death, and significantly downregulates cell signaling pathways in primary mediastinal B-cell lymphoma (PMBL): Obinutuzumab may be a future potential targeted agent for treatment of PMBL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3096. doi:10.1158/1538-7445.AM2014-3096

P13: Vitamin D3 therapy increases cryoablation efficacy: A novel strategy for the treatment of prostate cancer

Introduction Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances prostate cancer cell death. Vitamin D 3 (VD 3 ) is a natural steroidal hormone that has been shown to exhibit cytotoxic properties preferentially inducing apoptosis in a variety of human cancer cells. Human prostate cancer cells are known to be resistant to many cytodestructive agents, including cryoablation and VD 3 . Here, we evaluated the efficacy of VD 3 combined with cryoablation on androgen insensitive human prostate cancer (PC-3 and LNCaP-HP) cell death. Methods Freezing and VD 3 exposure to PC-3 and LNCaP-HP cells were performed using both 2D and 3D culture systems and efficacy was determined by cell viability assays. Resultant cell death and specific signaling components were determined using apoptotic inhibitors, fluorescence microscopy, protease activity assays and immunoblotting. Results Exposure of LNCaP-HP cells to freezing (-15°C) or VD 3 (50 μM) results in minimal cell death (15% and 10% respectively 3 days post treatment), while a complete loss of viability was observed with the combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a decrease in pro-caspase-9 and -3 between 6 and 18 h post-exposure. Caspase activation assays confirmed the reduction in pro-caspase levels was a result of caspase activation. Protease inhibitors were incorporated into the combination protocol to determine the overall contribution of caspase activity in cell death. Inhibition of caspase-9 significantly blocked the combination induced cell death compared to cells that did not receive the inhibitor (55% vs. 21% viable, respectively). The addition of the caspase-8 inhibitor resulted in only minimal protection, indicating a specific mitochondrial-mediated event. Importantly, the combination was not effective when applied to normal prostate cells. Conclusions VD 3 sensitizes CaP cells to cryoablation. The significant increase in cell death was attributed to the activation of apoptosis, specifically through mitochondrial -mediated events. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.

A36 Session 10: Adjunctive Therapeutic Approaches: Aquaporins: potential targets to enhance cryoablation

Aquaporins have been implicated in the angiogenesis of cancer cells whereby most cancer cells exhibit an increase in aquaporins. There is recent research activity to suggest that employing aquaporin channel blockers can slow the progress of cancer angiogenesis. A recent study by our group showed that increasing the number of aquaporin at the cell surface improved viability of the cells following a cryopreservation protocol. The thinking behind this study was that increasing the availability of aquaporins would improve water movement during freezing thus reducing the availability of water for intracellular ice formation leading to improved viability. In effect, the study attempted to manipulate and broaden the inverse–U curve of cell survival. Conversely, to enhance the effect of cryosurgery this group attempted to flatten the inverse–U curve by blocking the aquaporins with mercuric chloride ( HgCl 2 ) . By preventing water movement during freezing, the cells would exhibit higher incidence of intracellular formation and thus increase cell death. The goal of this study was to determine whether blocking aquaporins would lead to increased cell death at slower freezing rates and higher end temperatures which are typically seen at the boundary of the ice ball toward the end of a cryosurgical procedure. Preliminary experiments at a freezing rate of 1 °C/min and a holding temperature of −10 °C or −20 °C for 1, 5, and 15 min show that HgCl 2 treated cells had significant increase in cell death after thawing and 24 h incubation. Although much more studies needs to be conducted, this approach may prove to enhance the effectiveness in treating cancer with cryosurgery.

Concentration-Dependent Bimodal Effect of Specific 18 kDa Translocator Protein (TSPO) Ligands on Cell Death Processes Induced by Ammonium Chloride: Potential Implications for Neuropathological Effects Due to Hyperammonemia

The role of the 18-kDa Translocator Protein (TSPO) in cell death induced by NH4Cl (1-50 mM) for 24-72 hours to human glioblastoma U118MG cells was investigated. Cell death was already observed after 48 hours of treatment with NH4Cl at 5 mM. Dose and time-responses curves indicated that 15 mM of NH4Cl applied for 72 hours was the optimal condition for our viability assays. For example, 72 hours of 15 mM of NH4Cl caused a 50.3% increase in propidium iodide uptake, and lactate dehydrogenase release was 41.2% of the positive control, indicating significant increases in cell death. Furthermore, compared to vehicle control, these experimental conditions resulted in a significant decrease of 44.9% of the mitochondrial activity, a 62.3% increase in incidence of collapse of mitochondrial membrane potential, and an increase of 49.0% of cardiolipin peroxidation. In addition, a significant 4.3 fold increase in the maximal binding capacity (Bmax) of TSPO was found in NH4Cl-exposed cells. Surprisingly, western blot analysis and real-time PCR did not demonstrate changes in TSPO expression. We also found that neither NH4Cl nor glutamine (a metabolic product of enhanced NH4Cl levels) inhibited binding of the TSPO ligand [(3)H]PK 11195. Interestingly, we observed a bimodal effect of the TSPO ligands PK 11195, Ro5-4864, and FGIN-1-27 on the toxicity of NH4Cl; such that 1-100 nM concentrations of TSPO ligands were protective, while concentrations above 1 μM enhanced NH4Cl-induced cell death processes. In conclusion, TSPO takes part in a bimodal way in the lethal effects induced by NH4Cl in glial type cells.

The reduced autophagic response by oxidative stress in angiotensin II-induced hypertrophic H9C2 cells causes more apoptotic cell death.

Autophagy is an important process in the pathogenesis of cardiovascular diseases, and angiotensin II (Ang II) plays a causative role in the induction of cardiomyocyte autophagy. The purpose of this study was to explore whether, under conditions of oxidative stress, levels and types of cell death were different in untreated and Ang II-treated cardiomyocytes (H9C2 cells). Treatment with 20 µM Ang II induced cardiac hypertrophy in H9C2 cells, with increased expression of the hypertrophic markers c-Fos, ß-myosin heavy chain, atrial natriuretic factor (ANF), and brain natriuretic factor (BNF). Under normal conditions, there was no difference in the levels of autophagic vacuoles and apoptotic bodies in untreated and Ang II-treated H9C2 cells. However, oxidative stress generated by 100 µM H₂O₂ triggered autophagy in untreated control cells, but had a reduced effect in Ang II-induced hypertrophic cells, resulting in more cell death, and this was associated with a decrease in connexin 43 expression. Blocking this autophagic response with 3-methyladenine resulted in a significant increase in cell death and apoptosis of H9C2 cells but did not significantly affect the response of Ang II-treated cells. The autophagic response to 100 µM H₂O₂ provides a survival advantage for cells and this is reduced by Ang II treatment.

Persistent Loss of Hippocampal Neurogenesis and Increased Cell Death following Adolescent, but Not Adult, Chronic Ethanol Exposure

Although adolescence is a common age to initiate alcohol consumption, the long-term consequences of exposure to alcohol at this time of considerable brain maturation are largely unknown. In studies utilizing rodents, behavioral evidence is beginning to emerge suggesting that the hippocampus may be persistently affected by repeated ethanol exposure during adolescence, but not by comparable alcohol exposure in adulthood. The purpose of this series of experiments was to explore a potential mechanism of hippocampal dysfunction in adults exposed to ethanol during adolescence. Given that disruption in adult neurogenesis has been reported to impair performance on tasks thought to be hippocampally related, we used immunohistochemistry to assess levels of doublecortin (DCX), an endogenous marker of immature neurons, in the dentate gyrus (DG) of the hippocampus 3-4 weeks after adolescent (postnatal day, PD28-48) or adult (PD70-90) intermittent ethanol exposure to 4 g/kg ethanol administered intragastrically. We also investigated another neurogenic niche, the subventricular zone (SVZ), to determine if the effects of ethanol exposure were region specific. Levels of cell proliferation and cell death were also examined in the DG via assessing Ki67 and cleaved caspase-3 immunoreactivity, respectively. Significantly less DCX was observed in the DG of adolescent (but not adult) ethanol-exposed animals about 4 weeks after exposure when these animals were compared to control age-mates. The effects of adolescent ethanol on DCX immunoreactivity were specific to the hippocampus, with no significant exposure effects emerging in the SVZ. In both the DG and the SVZ there was a significant age-related decline in neurogenesis as indexed by DCX. The persistent effect of adolescent ethanol exposure on reduced DCX in the DG appears to be related to significant increases in cell death, with significantly more cleaved caspase-3-positive immunoreactivity observed in the adolescent ethanol group compared to controls, but no alterations in cell proliferation when indexed by Ki67. These results suggest that a history of adolescent ethanol exposure results in lowered levels of differentiating neurons, probably due at least in part to increased cell death of immature neurons. These effects were evident in adulthood, weeks following termination of the chronic exposure, and may contribute to previously reported behavioral deficits on hippocampal-related tasks after chronic ethanol exposure in adolescence.

Neuronal degeneration, synaptic defects, and behavioral abnormalities in tau45-230 transgenic mice

The complement of mechanisms underlying tau pathology in neurodegenerative disorders has yet to be elucidated. Among these mechanisms, abnormal tau phosphorylation has received the most attention because neurofibrillary tangles present in Alzheimer’s disease (AD) and related disorders known as tauopathies are composed of hyperphosphorylated forms of this microtubule-associated protein. More recently, we showed that calpain-mediated cleavage leading to the generation of the 17kDa tau45-230 fragment is a conserved mechanism in these diseases. To obtain insights into the role of this fragment in neurodegeneration, we generated transgenic mice that express tau45-230 and characterized their phenotype. Our results showed a significant increase in cell death in the hippocampal pyramidal cell layer of transgenic tau45-230 mice when compared to wild-type controls. In addition, significant synapse loss was detected as early as six months after birth in transgenic hippocampal neurons. These synaptic changes were accompanied by alterations in the expression of the N-methyl-d-aspartate glutamate (NMDA) receptor subunits. Furthermore, functional abnormalities were detected in the transgenic mice using Morris Water Maze and fear conditioning tests. These results suggest that the accumulation of tau45-230 is responsible, at least in part, for neuronal degeneration and some behavioral changes in AD and other tauopathies. Collectively, these data provide the first direct evidence of the toxic effects of a tau fragment biologically produced in the context of these diseases in vertebrate neurons that develop in situ.

High Mechanical Strain of Intervertebral Disc Cells Promotes Secretion of Inflammatory Factors Associated with Disc Degeneration and Pain

IntroductionLow back pain is a debilitating disorder affecting a multitude of people and is currently ranked sixth among costliest treatable chronic illnesses.1 Low back pain is often attributed to...